1. Field of the Invention
The invention relates to the field of recombinant DNA technology for the production of chondroitin, including the production of chondroitin sulfate via a combination of recombinant bacterial fermentation and post-fermentation sulfation.
2. Background Art
Chondroitin belongs to a family of heteropolysaccharides called glycosaminoglycans (GAGs). Glycosaminoglycans (GAGs) are unbranched, negatively charged polysaccharide chains composed of repeating disaccharide units, one of which is an acidic sugar and the other of which is an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), that can be sulfated. Because of their inflexible nature and high negative charge, GAGs exhibit highly extended conformations occupying large amounts of space, attracting cations and water and forming porous gels in the extracellular matrix. As such, GAGs, which are found in most animals, help to hydrate and expand tissues and enable the matrix to withstand compressive forces. The cartilage matrix lining the knee joint, for example, can support pressures of hundreds of atmospheres by this mechanism.
Chondroitin sulfate is important for maintenance of cartilage strength and resilience and is marketed as a nutritional supplement to reduce joint pain and promote healthy cartilage and joint function. Clinical studies support the use of chondroitin sulfate for treatment of osteoarthritis (See, for example, Kahan et al., Arthritis and Rheumatism 2009; 60:524-533; Michel et al., Arthritis and Rheumatism 2005; 52:779-786 and Uebelhardt et al., Osteoarthritis and Cartilage 2004; 12:269-276), interstitial cystitis (See, for example, Nickel et. al., BJU Int. 2009; 103:56-60 and Cervigni et al., Int. Urogvnecol. J. Pelvic Floor Dysfunct. 2008; 19:943-947), and synovitis (See, for example, Hochberg and Clegg, Osteoarthritis and Cartilage 2008; 16(Suppl. 3):S22-S24 and Möller, Osteoarthritis and Cartilage 2009; 17(Suppl. 1):S32-S33). These documents are incorporated by reference herein in there entireties.
Chondroitin sulfate is currently produced by extraction from cartilage of animals including cows, pigs, sharks and poultry, using chemical and enzymatic treatments to dissociate the polysaccharide from the protein and produce a polysaccharide product of varying quality (Barnhill et al., J. Am. Pharm. Assoc. 2006; 46:14-24, Volpi, J. Pharm. Pharmacol. 2009; 61:1271-1280).
Chondroitin contains D-glucuronic acid (GlcUA) and N-acetyl-D-galactosamine (GalNAc). It is composed of a disaccharide repeating unit β3GalNAc-β4GlcUA. Typically, the GalNAc residues are variably sulfated at the 4 and 6 positions. Chondroitin sulfate occurs naturally as a component of proteoglycans that are structural components of cartilaginous tissue, such as joints, in humans and other animals. Proteoglycans consist of a core protein and a polysaccharide component, such as chondroitin sulfate, which is covalently attached to the protein through an oligosaccharide linker as shown in FIG. 1. The core protein is decorated with multiple polysaccharide chains. Proteoglycans can be anchored in the cell membrane with the polysaccharide-containing portion of the protein present in the extracellular space or can be secreted and localized in the extracellular matrix (Prydz and Dalen, J. Cell Sci. 2000; 113:193-205).
The glycosyltransferase enzymes responsible for synthesizing the chondroitin backbone (chondroitin synthases) do so by adding alternating monosaccharide units of GalNAc and GlcUA from UDP-GalNAc and UDP-GlcUA donors to an accepting substrate. Theses enzymes have been identified in humans (Kitagawa et al., J. Biol. Chem. 2001; 276:43894-43900; Yada et al., J. Biol. Chem. 2003; 278:39711-39725), and homologs of human chondroitin synthase have been identified in a variety of other animals including horse, cow, rodents, dog, chicken, zebra fish, nematodes, and insects (www.ncbi.nlm.nih.gov/homologene/8950).
Some bacteria also produce chondroitin or chondroitin-like polysaccharide polymers as a component of their capsule. Unlike the chondroitin sulfate found in vertebrates, microbial chondroitin is not present as a proteoglycan, but rather as a lipid-linked polysaccharide on the bacterial cell surface and as free (i.e., not cell-associated) polysaccharide in culture media (Whitfield, Annu. Rev. Biochem. 2006; 75:39-68; DeAngelis, Glycobiol. 2002; 12:9R-16R).
Two bacteria, E. coli K4 (Rodriguez et al., Eur. J. Biochem. 1988; 177:117-124) and Pasteurella multocida serotype F (Rimler, Vet. Rec. 1994; 134:191-192), were reported to produce non-sulfated, chondroitin-like, capsular polysaccharides that potentially could be chemically modified to produce chondroitin sulfate. E. coli K4 was shown by Rodriguez et al. to produce an unsulfated chondroitin backbone with fructose side branches (K4 antigen) as a capsular polymer component. Ninomiya et al. (J. Biol. Chem. 2002; 277:21567-21575) identified and sequenced key genes required for biosynthesis of the chondroitin-like capsular polysaccharide in E. coli K4. These sequences were deposited with GenBank™ having accession number AB079602. The sequences disclosed by Ninomiya et al. comprise the so-called “region 2” portion of the “group 2” capsule gene cluster of E. coli K4. A detailed description of the organization of capsule gene clusters in E. coli is provided by Whitfield (Annu. Rev. Biochem. 2006; 75:39-68). The region 2 genes of E. coli group 2 capsule gene clusters encode the proteins that determine the structure of the capsular polysaccharide. The AB079602 sequence includes a gene, termed kfoC, that encodes the E. coli K4 chondroitin polymerase. The E. coli K4 chondroitin polymerase is a bifunctional glycosyltransferase that transfers GlcUA and GalNAc alternately to the non-reducing end of a chondroitin saccharide chain and related oligosaccharides, producing the chondroitin backbone of the K4 antigen polysaccharide. The chondroitin-like capsule polysaccharide produced by E. coli K4 contains fructose, linked (β1,3) to the GlcUA residues of chondroitin. Pasteurella multocida Type F also produces an unsulfated chondroitin capsule component and the glycosyltransferase responsible for chondroitin polymerization in this organism has also been cloned, as reported in DeAngelis & Padgett-McCue, J. Biol. Chem. 2000; 275:24124-29. Like the K4 chondroitin polymerase, the Pasteurella chondroitin synthase (pmCS, Genbank Accession No. AAF97500) is a single polypeptide enzyme that can synthesize a chondroitin polymer from UDP-GlcUA and UDP-GalNAc when provided with an appropriate acceptor substrate.
Traditional methods of chondroitin sulfate production involving purification from animal tissue can be laborious and cost intensive. Moreover, production of chondroitin sulfate from animal tissue is necessarily associated with the potential for infectious agents to be present in the chondroitin sulfate products. Care must be taken during production from animal tissues to minimize the likelihood of such potential infectious agents. Such shortcomings can be addressed by using alternative approaches utilizing recombinant DNA technology for production of chondroitin. Recently, microbial production of chondroitin has been suggested by DeAngelis (US Patent Application Publication No. 20030109693) and by Cimini et al. (Appl. Microbiol. Biotechnol. 2010; 85(6):1779-87 (Epub Oct. 1, 2009)). However, the known microorganisms that produce chondroitin (Pasteurella multocida) or chondroitin-like (E. coli K4) polysaccharides are known pathogens to various mammals and therefore unsuitable for large scale fermentation. They are also relatively low producers of the polysaccharide.
In particular, P. multocida is considered not suitable for commercial production of chondroitin because of its low yield, requirement of expensive media, and Biohazard Level 2 (BL2) status, which requires specialized and expensive facilities. High yields from a microorganism would be necessary for commercially profitable production of chondroitin. DeAngelis (US Patent Application Publication No. 20030109693) mentions the possibility of expressing pmCS in host cells such as a food grade Lactococcus or Bacillus to synthesize recombinant chondroitin. However, Bacillus is a gram positive bacterium and, as such, has a very different membrane/cell wall structure than gram negative organisms like E. coli and Pasteurella multocida. Efficient secretion of the polymer would, therefore, be expected to be problematic in Bacillus. 
E. coli K4 is also unsuitable for production of chondroitin because it is known to be a human pathogen. Moreover, it does not produce chondroitin per se, but instead produces, as noted above, a fructosylated form of chondroitin. Extensive chemical or enzymatic modification of this polysaccharide is required to produce chondroitin. Such modification increases the total cost of the process. Additionally, it requires introduction of further processes and quality control measures to determine that such modification was complete and generated a consistent product.
There is a need, therefore, for an efficient, safe and cost effective process for the production of chondroitin. The present invention addresses this need by providing constructs and host cells and methods for recombinant microbial production of chondroitin which can subsequently be sulfated to produce chondroitin sulfate.